1. Field of the Invention
The present invention relates to a rapid method for detecting microorganisms by means of a fluorescence analysis method using umbelliferone derivative.
2. Description of prior art
It has been socially demanded to keep food and beverage free from microbial contamination, and it is provided by Japanese law that the food such as edible meat, soft drink and fish paste product should not contain any coliform organisms.
Therefore, the sanitary quality of such a product has to be controlled strictly in a process of manufacture and in a course of circulation for sale.
It is very important for this purpose that a microbial inspection be carried out rapidly and the result concerning microbial contamination be obtained as soon as possible.
Known methods for detecting microotganisms, however, require at least 24 hours incubation period for detection. As the conventional methods for detecting microorganisms, the Plate Count Method and the Most Probable Number Method are known (Official Methods of Analysis of the Association of Official Analytical Chemists, Official First Action, Eleventh Edition, page 843, 1970, U.S.A.), as follows: Plate Count Method (suitable for analyzing foods in which large number of microbes are expected.)
Sample (food) is homogenized and diluted with sterile water. Each decimal dilution of 10.sup.-1 to 10.sup.-4 of the homogenate is poured into a petri dish together with a nutrient agar medium. Then the dishes are incubated for 24 to 48 hours and the number of colonies grown on a plate is counted, on basis of which plate count per gram is determined. Most Probable Number (MPN) method (recommended for a use in a routine surveillance of foods in which a small number of microbes, especially coliform bacteria, may be expected.) The dilution of the homogenate is transferred into 3 to 5 fermentation tubes containing nutrient medium such as lactose bouillon medium and Brilliant Green Lactose Bie (BGL B) and the tubes are incubated for 24 to 48 hours. Then the number of tubes showing gas production is counted (positive presumptive test). Afterwards the confirmed test for coliform bacteria, on all positive presumptive tests, is conducted.
MPN is calculated using MPN Table on basis of the number of tubes showing gas production.
In either case, microorganisms in a nutrient medium have to be cultured till the growth of the microorganisms can be measured with the naked eye.
Accordingly, these common methods require at least 24 hours incubation period for detection and the product manufactured has to be stored till the result is obtained. Recently, a number of papers concerning rapid methods for detecting microorganisms have been published.
In these methods, changes of an impedance or, PH of culture medium, or the amount of consumed oxygen, or generated carbon dioxide gas, accompanied with the growth of microorganisms is measured and the number of microorganisms is determined based on the relationship between the resultant values and the number of microorganisms. However, these methods are accessible only for the inspection of a sample solution containing more than 10.sup.5 /ml microorganisms.
Additionally, these values may be affected by coexisting materials, and these rapid methods are not satisfactory for practical use owing to their low accuracy and limited range of detection.